Laccase is an enzyme that is a blue copper oxidase. It is believed to have been first obtained from the Japanese tree Rhus venicifera. Yshida, H., Zur Chemie des urushi-Firniss, J. Chem. Soc. (Tokyo) 43, 472, 1883. It has since been found in a number of higher plants, but not at high levels. The main source of laccase for commercial purposes is fungi. White rot fungi Phanerochaete chrysosporium and Trametes (Coriolus) versicolor are common commercial sources of the enzyme. Other fungi producing laccase include Polyporus, Pleurotus, Philota, Neurospora, Podospora and Aspergillus.
The enzyme has a number of uses. Examples include catalyzing the oxidation of compounds such as o,p-diphenols, aminophenols, polyphenols, polyamines and inorganic ions. (See, e.g. Yaropolov et al., xe2x80x9cLaccase Properties, Catalytic Mechanism, and Applicabilityxe2x80x9d Applied Biochemistry and Biotechnology 49:257-280 (1994)). Its"" use as a marker enzyme in enzyme immunoassay (EIA) has also been discussed, as well as its"" use in oxidation of steroids and synthesis of vinblastine, a cytostatic compound used in treating malignant diseases.
The most common use of laccase, however, is in connection with the paper and pulp industry. Lignin is a rigid organic polymer and harsh physicochemical conditions must be used to attack or modify the substance. One answer in the search for means to break down lignin was found in the white rot fungi which can naturally destroy lignin, using laccase. In plants, laccase is localized in woody tissues and cell walls of herbaceous species and it is believed it participates in lignin biosynthesis. It is involved in breaking down lignin as well as creating lignin polymers. It is also especially useful as a xe2x80x9cbiological gluexe2x80x9d when manufacturing glued wood products. Such products include construction and industrial plywood, oriented strand board, particleboard and medium density fiberboard.
Currently, the adhesive used is either a urea-formaldehyde type or a phenol-formaldehyde resin. There are disadvantages associated with use of formaldehyde in producing such products. Processing and end use monitoring are required as the levels of formaldehyde cannot exceed certain controls. Thus, there has been considerable interest in using such natural alternatives as laccase. It is reported that more than 1.2 million metric tons of adhesive resin solids are used to bond glued wood products in the United States. Which adhesive is used is driven by cost per unit of production, process compatibility and end-use durability. See, xe2x80x9cTechnical and Market Opportunities for Glued Wood Productsxe2x80x9d Adhesive Age May 31, 1996 V39, N6 p.609.
An example of such a process is described by Kharazipour et al in U.S. Pat. No. 5,505,772 and by Olesen et al. at U.S. Pat. No. 5,618,482. In general, fibers and chips from wood or wood-like materials are defibrated by mechanical, steam, or other process. Laccase is then brought into contact with the material in a solution which may contain various auxiliary elements. Since laccase is a large molecule, a mediator may be utilized to aid the enzyme in penetrating the wood and may be added to the solution. The mix is incubated and may then be shaped into formed boards.
A problem with using laccase produced by white-rot fungi, however, is that it is produced in relatively low amounts. Saloheimo, M. and Niku-Paavola, M-L. xe2x80x9cHeterologous Production of a Ligninolytic Enzyme: Expression of the Phlebia radiata Laccase gene in Trichoderma reeseixe2x80x9d Bio/Technology 9:987-990 (1991). Native expression is described at about 10,000 U/liter from Trametes versicolor after induction, which is about 220-250 mg/liter if all was laccase I, and about 65-75 mg/liter if the laccase was laccase II. Bourbonnais et al. Appl. Environ. Microb. 61, no.5 pp.1876-1880 (May 1995). Other experiments have shown expression in Myceliophthora thermophila and in Aspergillus oryzae was at about 5 mg/liter. Measurements by the inventors reflect that the natural laccase expression in plants is about less than 0.001% of soluble plant protein. Attempts by Saloheimo and Niku-Paavola to improve on these levels by using heterologous expression yielded 20 mg/l secreted active laccase. Berka et al. noted that expression levels are too low for commercial purposes. Berka et al. Applied and Environmental Microbiology p.3151-3157 (August 1997). While others have attempted to introduce laccase-encoding nucleotide sequences into plants for the purpose of changing the lignin content of the plant in WO 98/11205 and WO97/45549, they do not teach production of laccase at commercially acceptable levels for extraction and use.
The inventors have discovered that it is possible to produce commercially acceptable quantities of laccase in plants. This results in a considerable decrease in cost of producing the enzyme. Thus it is an object of the invention to produce laccase in plants at commercially useful levels.
Finally, also provided are improvements on methods of transforming these and other plants using Agrobacterium. Modifications to selection of the bacterial strain used, and of processing the strain are provided.
These and further objectives will become apparent from the description.
All references cited herein are incorporated herein by reference.
Plants and a process of using them is described in which commercial levels of laccase are produced in plants. Improvements to Agrobacterium-mediated transformation processes are also provided.